Organoids are in vitro three-dimensional(3D)multicellular cultures that are generated through deploying the self-renewal and self-organizing capacities of stem cells.They recapitulate key structural and functional features of corresponding organs or tissues,providing an ideal in vitro model and research platform for the study of developmental biology,regenerative medicine,disease modeling and drug development.The conventional organoid culture system mainly relies on manual operations with lengthy and complicated procedures,which generate organoid cultures of individual variations and batch differences,limiting their translational applications.Therefore,to engineer the organoid culture system by introducing microfluidic chip technology to enhance the throughput and automation level,is of great significance for achieving large-scale,homogeneous,and standardized organoid cultures.This article reviews the current research progress of high-throughput and automated organoid chips and discusses the main limitations and potential challenges for the future study.

Objective To explore novel methods for efficient respiratory viral infection of organoids by microinjection and polarity inversion techniques.Methods Lung tissue samples were obtained from 8-week-old male C57BL/6 mouse,and respiratory epithelial cells were extracted to establish a transwell organoid culture model.The green fluorescent protein(GFP)labeled influenza virus PR8(GFP-PR8)was quantitatively injected into organoids by improving the traditional microinjection platform,and morphologic changes in organoids and the immunofluorescence staining characteristics of tight junction proteins and microtubule proteins were observed.Polarity inversion apical-out(AO)was induced by suspension culture,and the morphological characteristics of polarity inversion was determined by HE staining.Normal and inverted organoids were infected with PR8,and the infection efficiency and expression differences of key pathway genes under different virus concentrations were observed.Results Ordinary organoids showed a significant increase in volume after microinjection.Following PR8 injection,the efficiency of infection was significantly higher in the apical region of organoids,accompanied by noticeable damage,as evidenced by significant down-regulation of tight junction proteins and microtubule protein expression.After suspension culture of the organoids,the polarity of ciliated cells gradually inverted outward over time,and the proportion of AO organoids stabilized on the 6th day.The efficiency of viral infection significantly increased in the inverted organoids,accompanied by significant cellular damage.After PR8 infection at 0.01 MOI,AO organoids showed significant changes in the inflammatory pathway and differentiation-related genes,with the opposite trend observed after higher concentration of PR8 infection.Conclusion Both polarity inversion and microinjection techniques significantly enhance the efficiency of influenza virus infection in organoids,thereby facilitating organoid widespread application in the field of respiratory tract infections.

Objective To explore the mechanism of Lkb1 regulated epithelial regeneration in asthma by airway organoid culture.Methods Lkb1f/f(the control group,n=10)and Scgb1a1CreER;Lkb1f/fmice(the Lkb1 knockout group,n=9)were taken to establish allergic asthma models by aerosol inhalation of ovalbumin(OVA).Bronchial lavage fluid(BALF)and lung tissue were collected.The number of inflammatory cells in BALF were counted.The amount of CLCA3 positive cells was compared by immunofluorescence staining of lung tissue sections.Club cells were selected by flow cytometry for organoid culture.The average diameter of organoids and organoid formation rate were calculated.Expression levels of goblet cell marker CLCA3,cilia cell markers FOXJ1 and AMPK in Club cells were detected by RT-PCR.Results There were no significant differences in the number of macrophages,eosinophils,neutrophils and lymphocytes in BALF between the control group and the Lkb1 knockout group.The number of CLCA3 positive cells were decreased after Lkb1 knockout.Results of organoid culture showed that the average diameter of organoids derived from Club cells and organoid formation rate were decreased after the absence of Lkb1.The expression of FOXJ1 was reduced.After Lkb1 deletion,the expression of AMPKα in Club cells were decreased and the proliferation of Club cells was inhibited.Activation of AMPK,the downstream signaling pathway of Lkb1,could attenuate the effect of Lkb1 deficiency on the regeneration of Club cells.Conclusion Lkb1 promotes the proliferation of airway progenitor cells by AMPK pathway.

Objective To establish in vitro the small intestinal organoid culture system and to investigate the effect of lipopolysaccharide(LPS)on the growth of small intestinal organoids and the secretion of inflammatory factors.Methods In vitro,the small intestinal crypt cell mass of C57BL/6 mice was aseptically isolated,collected and embedded in organoid matrix.Under the support of complete medium,the small intestinal organoids with three-dimensional multi-leaf structure with small intestinal epithelioid structure were formed.The small intestinal organoids were subcultured after 5-7 d culture.On the third day after passage,the small intestinal organoids were randomly divided into different mass concentrations of LPS groups(0,150,175,200,225,250,275 and 300 mg/L).After 24 h and 48 h of LPS induction,morphological changes of small intestinal organoid growth and differentiation were observed.CCK-8 method was used to detect the effect of different time points and mass concentrations of LPS on the proliferative activity of small intestinal organoids after induction of inflammation.The effects of four different mass concentrations of LPS(0,175,200 and 225 mg/L)on expression levels of granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin(IL)-1α,IL-6 and IL-10 in organoid culture supernatant at different times were detected by enzyme-linked immunosorbent assay(ELISA).Results The mouse small intestinal organoid culture system was preliminarily constructed.After different time and mass concentration of LPS induced inflammation of small intestinal organoids,it was observed by morphology that small intestinal organoids would have different degrees of expansion and apoptosis in lumen.The proliferation,differentiation and budding of damaged intestinal epithelial crypts or intestinal stem cells were also inhibited to varying degrees,indicating that the growth of small intestinal organoids would be limited to varying degrees after induced inflammation.The proliferation activity of small intestinal organoids decreased to varying degrees after 24 h and 48 h of LPS induction at 175-225 mg/L(P＜0.05),but the cell viability was still greater than 50%.The levels of IL-1α,IL-6 and GM-CSF partially increased after induction with 200 mg/L and 225 mg/L LPS for 24 h and 48 h(P＜0.05).The level of IL-10 decreased after induction with 200 mg/L LPS for 24 h and 48 h(P＜0.05).Conclusion In this study,a model of intestinal inflammatory injury in vitro induced by LPS with different mass concentrations and time points is preliminarily constructed,which provides a more reliable research platform for the mechanism research of intestinal diseases and the screening of effective drugs in the future.

Objective To establish a culture method for micropapillary lung adenocarcinoma organoids and conduct targeted drug screening.Methods Organoids were extracted and cultured from a surgical tissue sample of a patient diagnosed with micropapillary lung adenocarcinoma,and the growth of lung cancer organoids was observed and recorded dynamically.The morphological and gene expression characteristics of tumor cells between lung cancer organoids and parental tissue were compared using hematoxylin eosin(HE)staining and immunohistochemical methods.Real time fluorescence quantitative polynucleotide chain reaction(qRT-PCR)method was used to detect gene mutations in lung cancer parental tissue and organoids.Finally,based on results of genetic testing,targeted drugs were selected and their therapeutic effects were verified.Results We have successfully cultured spherical organoids from micropapillary lung adenocarcinoma tissue,which can be passaged for at least 3 generations.HE staining results showed that the morphology of tumor cells in organoids was roughly consistent with that of parental tissue.The immunohistochemical results showed that the protein expression levels of various genes in lung cancer organoids and parental tissue were roughly the same.Results of gene mutation analysis showed that the mutated genes in lung cancer parental tissue and organoids were consistent,both reflecting RET fusion.The screening results of targeted drugs based on lung cancer organoids showed that vandertinib had the best anti-tumor effect in vitro.Conclusion Drug screening experiments based on micropapillary lung adenocarcinoma organoids can screen highly efficient targeted drugs in a short period of time,which may benefit patients with micropapillary lung adenocarcinoma.

Organoids are the three-dimensional culture of adult stem cells or pluripotent stem cells in vitro to form tissue analogs with specific structures,which have highly similar tissue properties and physiological functions to the corresponding organs.The emergence of organoid technology has laid an important foundation for research in organ development,disease modeling and drug discovery.Tumor organoid,as an important branch of organoids,is a transition between cell lines and animal models,which can well retain the histological and mutational characteristics of tumors in patients and play an essential role in building tumor organoid sample libraries,reconstructing tumor microenvironment,studying the tumor development mechanism as well as formulating personalized treatment plans and drug screening.Tumor organoids help clinicians to realize precise treatment for patients.However,some factors still limit the further development of tumor organoids,such as the lack of microenvironmental components,vascular structure,high culture cost and technical difficulties.In this review,we summarize the applications and challenges of organoid technology in basic tumor research and clinical translation and look forward to the future development of tumor organoids.

Organoids are in vitro 3D tissue cultures that retain the characteristics of their source tissue and organs,and have been widely used in biomedical fields.Organoids have emerged as effective models for investigating diseases,contribute to further understanding of pathophysiology and molecular mechanisms of diseases,and provide technical support for personalized treatment of genetic diseases.This article specifically explores the application of organoid technology in childhood genetic diseases.It emphasizes the advantages and potential advancements of this technology in disease modeling,correction of genetic defects and personalized treatment,thereby serving as a valuable reference for future research endeavors.

Cerebral organoids are three-dimensional nerve cultures induced by embryonic stem cells(ESCs)or induced pluripotent stem cells(iPSCs)that mimic the structure and function of human brain.With the continuous optimization of cerebral organoid culture technology and the combination with emerging technologies such as organ transplantation,gene editing and organoids-on-chip,complex brain tissue structures such as functional vascular structures and neural circuits have been produced,which provides new methods and ideas for studying human brain development and diseases.This article reviews the latest advances in brain organoid technology,describes its application in neurological diseases and advances in stroke modeling and transplantation treatment.

Objective To construct an in vitro"metabolic memory"cell model of HT-22 mouse hippocampal neurons induced by high glucose,and to investigate the effect of"metabolic memory"on apoptosis and histone acetylation in HT-22 cells.Methods HT-22 cells were cultured in high glucose medium(glucose concentration was 55 mmol/L)and conventional glucose medium(glucose concentration was 25 mmol/L),and cells were divided into the control group(NG 4,6 and 8 groups,25 mmol/L glucose was cultured for 4,6 and 8 days,respectively),the high glucose group(HG 4,6 and 8 groups,respectively)and the metabolic memory group(HG2NG2,HG2NG4,HG2NG6,HG4NG2 and HG4NG4 groups,high glucose culture for 2 days to 25 mmol/L glucose culture for 2,4 or 6 days,high glucose culture for 4 days to 25 mmol/L glucose culture for 2 or 4 days).Cell viability was detected by CCK-8 method.The release of lactate dehydrogenase(LDH)in cell culture supernatant was detected,and the optimal time to establish a"metabolic memory"model was selected.Subsequently,cells were divided into the NG4 group,the NG8 group,the HG4 group,the HG4NG4 group and the HG8 group,and the cell morphology of each group was observed by optical microscope.The apoptosis rate was detected by flow cytometry.The activities of deacetylase(HDAC)and histone acetyltransferase(HAT)were detected by enzyme-linked immunosorbent assay(ELISA).Western blot assay was used to detect expression levels of histone deacetylase 4(HDAC4),B lymphocyte tumor 2(Bcl-2),Bcl-2 related X protein(Bax)and Caspase-3 protein.Results The HG4NG4 group was the ideal cell model with high glucose metabolic memory.Cells of the NG4 group and the NG8 group were interwoven into a dense network,growing well,with spindle shaped cells and distinct synaptic structures.However,in the HG4 group and the HG8 group,the cell body became round,synaptic structure disappeared and growth was inhibited.In the HG4NG4 group,the number of cells increased but their morphology was damaged.Results of flow cytometry showed that compared with the NG8 group,the apoptosis rates were significantly increased in the HG8 group and the HG4NG4 group(P＜0.05).ELISA results showed that compared with the NG8 group,the expression levels of HDAC4,Bax,and Caspase-3 proteins increased in the HG8 group and the HG4NG4 group,while the expression level of Bcl-2 protein significantly decreased(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels of HAT and HDAC in the HG4NG4 group.Western blot reslts showed that compared with the NG8 group,the levels of HDAC4,Bax and Caspase-3 protein increased in the HG8 group and the HG4NG4 group(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels in the HG4NG4 group.Conclusion HT-22 mouse hippocampal neurons cultured with 55mmol/L high glucose for 4 days,and then cultured with 25 mmol/L glucose for 4 days are the ideal"metabolic memory"cell model.The mechanism may be related to the increased activity of HDAC,HAT and HDAC4 expression in the hyperglycemic model.

Objective To explore the effect and mechanism of anti-mesangial cell-proliferation-peptide 2(AMPP2)on mesangial cell proliferation induced by transforming growth factor β1(TGF-β1).Methods Mesangial cells were cultured in vitro and treated with TGF-β1(10 μg/L)and AMPP2(10 ng/L).According to different intervention factors,mesangial cells were divided into four groups:the control group,the AMPP2 group,the TGF-β1 group and the TGF-β1+AMPP2 group.The proliferation activity of mesangial cells was detected by CCK-8.The relative protein expression of cyclin dependent kinase 4(CDK-4),cyclin dependent kinase 6(CDK-6),proliferating cell nuclear antigen(PCNA),α-smooth muscle actin(α-SMA),collagen-Ⅰ(COL-Ⅰ)and fibronectin(FN)were examined by Western blot assay.The relative mRNA expression of α-SMA,COL-Ⅰ and FN were detected by qPCR.Results Compared with the control group,proliferation activity of mesangial cells was significantly increased in the TGF-β1 group(P＜0.05).The proliferation activity of mesangial cells was markedly decreased in the TGF-β1+AMPP2 group compared with that of the TGF-β1 group(P＜0.05).Compared with the control group,protein levels of CDK-4,CDK-6,PCNA,α-SMA,COL-Ⅰand FN in cells were significantly increased in the TGF-β1 group(P＜0.05),as well as the mRNA levels of α-SMA,COL-Ⅰand FN(P＜0.05).In the TGF-β1+AMPP2 group,the protein and mRNA levels of α-SMA,COL-Ⅰand FN and the protein levels of CDK-4,CDK-6 and PCNA were markedly decreased compared with those of the TGF-β1 group(P＜0.05).Compared with the control group,levels of p-SMAD3/SMAD3 was remarkably upregulated in the TGF-β1 group(P＜0.05),while levels of p-SMAD3/SMAD3 was remarkably downregulated in the TGF-β1+AMPP2 group compared with those of the TGF-β1 group(P＜0.05).Conclusion AMPP2 may inhibit mesangial cell proliferation by regulating TGF-β1/SMAD3 pathway.